Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate fetal development of protein C, a pregnant ovine model was used. Protein C was isolated from ovine plasma, and a polyclonal antibody was raised. Citrated plasma was obtained from undisturbed chronically catheterized fetal lambs. On Western blot, nonreduced adult ovine protein C had a molecular mass of 70 kD. Fetal ovine protein C was determined to have a molecular mass of 4 to 6 kD larger than the adult molecule. Crossed immunoelectrophoresis demonstrated slightly increased anodal migration of the fetal form. Isoelectric focusing demonstrated a decreased pI of the fetal molecule (4.45 versus 4.6). The ovine protein C molecules were deglycosylated with N-glycanase. Deglycosylated fetal protein C migrated more similarly to the adult form, although a portion of the fetal form persisted. These experiments demonstrate the first example of a unique fetal form of a vitamin K-dependent protein and are compatible with increased glycosylation of fetal ovine protein C. It is speculated that altered posttranslational processing may exist as a general process by which certain coagulation proteins are modified during fetal development. mRNA was isolated from maternal and fetal hepatic tissue and analyzed by Northern hybridization. Fetal plasma concentration and hepatic mRNA for protein C were both 40% of normal maternal values from midgestation onward. At term, protein C mRNA increased to adult range (p < 0.025), although plasma protein C concentration decreased slightly (p < 0.001). A transition from fetal to adult protein C form was found beginning 6 d before term birth, with a doubling time of 24 h. These data are compatible with a gestationally determined maturation of ovine protein C.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of a unique form of protein C in the ovine fetus: developmentally linked transition to the adult form. 778 46

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52kDa by SDS-PAGE analysis and 48,036Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8-12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 degrees C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl-l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10(4)-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1 microM(-1)s(-1). The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 degrees C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.
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PMID:BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility. 1743 97